Actinobacillus (formerly Haemophilus) pleuropneumoniae is a highly infectious porcine respiratory tract pathogen that causes porcine pleuropneumonia. Infected animals develop acute fibrinous pneumonia which leads to death or chronic lung lesions and reduced growth rates. Infection is transmitted by contact or aerosol and the morbidity in susceptible groups can approach 100%. Persistence of the pathogen in clinically healthy pigs also poses a constant threat of transmitting disease to previously uninfected herds.
The rapid onset and severity of the disease often causes losses before antibiotic therapy can become effective. Presently available vaccines are generally composed of chemically inactivated bacteria combined with oil adjuvants. However, whole cell bacterins and surface protein extracts often contain immunosuppressive components which make pigs more susceptible to infection. Furthermore, these vaccines may reduce mortality but do not reduce the number of chronic carriers in a herd.
There are at least 12 recognized serotypes of A. pleuropneumoniae with the most common in North America being serotypes 1, 5 and 7. Differences among serotypes generally coincide with variations in the electrophoretic mobility of outer membrane proteins and enzymes thus indicating a clonal origin of isolates from the same serotype. This antigenic variety has made the development of a successful vaccination strategy difficult. Protection after parenteral immunization with a killed bacterin or cell free extract is generally serotype specific and does not prevent chronic or latent infection. Higgins, R., et al., Can. Vet. J. (1985) 26:86-89; Macinnes, J. I. and Rosendal, S., Infect. Immun. (1987) 55:1626-1634. Thus, it would be useful to develop vaccines which protect against both death and chronicity and do not have immunosuppressive properties. One method by which this may be accomplished is to develop subunit vaccines composed of specific proteins in pure or semipure form.
A. pleuropneumoniae strains produce several cytolysins. See, e.g. Rycroft, A. N., et al., J. Gen. Microbiol. (1991) 137:561-568 (describing a 120 kDa cytolysin from A. pleuropneumoniae); Chang, Y. F., et al., DNA (1989) 8:635-647 (describing a cytolysin isolated from A. pleuropneumoniae serotype 5); Kamp, E. M., et al., Abstr. CRWAD (1990) 1990:270 (describing the presence of 103, 105 and 120 kDa cytolysins in A. pleuropneumoniae strains) and Welch, R. A., Mol. Microbiol. (1991) 5:521-528 (reviewing cytolysins of gram negative bacteria including cytolysins from A. pleuropneumoniae). One of these cytolysins appears to be homologous to the alphahemolysin of E. coli and another to the leukotoxin of Pasturella haemolytica. Welch, R. A., Mol. Microbiol. (1991) 5:521-528. These proteins have a molecular mass of approximately 105 kDa and are protective in mouse and pig animal models against challenge with the homologous serotype. However, cross-serotype protection is limited at best (Higgins, R., et al., Can. J. Vet. (1985) 26:86-89; Macinnes, J. I., et al., Infect. Immun. (1987) 55:1626-1634. The genes for two of these proteins have been cloned and expressed in E. coli and their nucleotide sequence determined. Chang, Y. F., et al., J. Bacteriol. (1991) 173:5151-5158 (describing the nucleotide sequence for an A. pleuropneumoniae serotype 5 cytolysin); and Frey, J., et al., Infect. Immun. (1991) 59:3026-3032 (describing the nucleotide sequence for an A. pleuropneumoniae serotype 1 cytolysin).
Transferrins are serum glycoproteins that function to transport iron from the intestine where it is absorbed, and liver, where it is stored, to other tissues of the body. Cell surface receptors bind ferrotransferrin (transferrin with iron) and the complex enters the cell by endocytosis. A. pleuropneumoniae, under iron restricted growth conditions, can use porcine transferrin as its sole iron source, but it cannot utilize bovine or human transferrin (Gonzalez, G. C., et al., Mol. Microbiol. (1990) 4:1173-1179; Morton, D. J., and Williams, P., J. Gen. Microgiol. (1990) 136:927-933). The ability of other microorganisms to bind and utilize transferrin as a sole iron source as well as the correlation between virulence and the ability to scavenge iron from the host has been shown (Archibald, F. S., and DeVoe, I. W., FEMS Microbiol. Lett. (1979) 6:159-162; Archibald, F. S., and DeVoe, I. W., Infect. Immun. (1980) 27:322-334; Herrington, D. A., and Sparling, F. P., Infect. Immun. (1985) 48:248-251; Weinberg, E. D., Microbiol. Rev. (1978) 42:45-66).
It has been found that A. pleuropneumoniae possesses several outer membrane proteins which are expressed only under iron limiting growth conditions (Deneer, H. G., and Potter, A. A., Infect. Immun. (1989) 57:798-804). Three of these proteins have been isolated from A. pleuropneumoniae serotypes 1, 2 and 7 using affinity chromatography. These proteins have molecular masses of 105, 76 and 56 kDa. (Gonzalez, G. C., et al., Mol. Microbiol. (1990) 4:1173-1179). The 105 and 56 kDa proteins have been designated porcine transferrin binding protein 1 (pTfBP1) and porcine transferrin binding protein 2 (pTfBP2), respectively. (Gonzalez, G. C., et al., Mol. Microbiol. (1990) 4:1173-1179). At least one of these proteins has been shown to bind porcine transferrin but not transferrin from other species (Gonzalez, G. C., et al., Mol. Microbiol. (1990) 4:1173-1179). It is likely that one of these proteins, either alone or in combination with other iron regulated outer membrane proteins, is involved in the transport of iron. The protective capacity of these proteins has not heretofore been demonstrated.